Total body tissue iron stores are an important diagnostic indicator to the health professional of the presence of diseased states or a susceptibility to diseased states in an individual.
Body iron is both a necessary component for cellular metabolism and a highly damaging agent when present in excess. For example, iron leashed to protein is an essential element in all cell metabolism and growth, but is toxic when unleashed (Herbert V., et al., Stem Cells, 92:1502-1509 (1994)). Because of its ability to switch back and forth between ferrous and ferric oxidation states, iron is both a strong biological oxidant and reductant. Ferric iron or Fe.sup.3+ is a relatively harmless form of iron. However, ferrous iron or Fe.sup.2+ plays a significant role in the generation of oxygen radicals, excess of which have been proven to be extremely harmful to the health of an individual.
The human diet contains a multitude of natural chemicals which are carcinogens and anti-carcinogens, many of which act by generating oxygen radicals, which initiate degenerative processes related to cancer, heart disease and aging (Ames, B., Science, 221:1256-1264 (1993)). Among these many dietary chemicals are many redox agents, including vitamin C and beta carotene.
Free radical damage is produced primarily by the hydroxyl radical (.OH). Most of the .OH generated in vivo comes from iron-dependent reduction of H.sub.2 O.sub.2 (Halliwell, B., Archives of Biochemistry and Biophysics, 246(2):501-514 (1986)), and supporting too much iron as a free radical-generating culprit leads to a risk of cancer. High total body tissue iron stores promotes diabetes, impotence, cancer, heart disease and liver disease (Stevens, R. G., et al., N. Engl. J. Med., 319:1047-1052 (1988).
When ferrous iron reduces H.sub.2 O.sub.2 to generate .OH, it becomes ferric iron. Vitamin C (ascorbic acid) converts ferric iron back to ferrous iron, itself becoming oxidized ascorbic acid, thus allowing another cycle of .OH generation from renewed ferrous iron (Aisen, P. et al., Int. Rev. Exp. Path, 31:1-46 (1990)).
The importance of iron in tumor growth is illustrated by the fact that iron deficiency slows the progression of malignancy (Hann, H. W. L. et al., Cancer Res., 48:4168-4174 (1988)). Catalytic iron free radical generation mutates DNA and promotes cancer. Conversely, too much catalytic iron within a cancer cell will destroy it.
Accordingly, it is a vital necessity to be able to determine the quantity of iron in the body. It is also important to have access to a non-invasive diagnostic assay which provides a quick and accurate way to determine total body tissue iron stores.
The body has developed a system to prevent against these undesirable effects of iron in the body.
Ferritin is an iron-containing spherical protein of 24 repeating subunits and a molecular weight of approximately 460,000. Ferritin exists in many different forms, all of which perform various roles relating to delivering, storing and controlling iron. Apoferritin contains no iron and is generated by the body in response to inflammation, disease or an iron challenge, for example, an iron overload.
In response to disease, cells of the reticulo-endothelial system manufacture a series of proteins called acute phase reactants, which aid in scavenging bacteria, viruses and any other foreign particles. Apoferritin is one of these acute phase reactants. The intracellular generation of apoferritin is a cytoprotective antioxidant stratagem of reticulo-endothelial cells (Balla, J. et al., Trans. Assoc. Am. Phys.., 105: (in press) (1993)). It is probably also a cytoprotective antioxidant stratagem of all proliferating cells, including cancer cells, since ferritin is elevated in acute leukemia and many other cancers, including solid tumors, particularly when metastatic (Voest, E. E., University of Utrecht, The Netherlands; June, 1993 (Thesis)). These serum ferritin levels may actually rise with chemotherapy. This hyperferritinemia is often mainly apoferritin generated in the tumor cells, but malignancy, like chronic inflammation, also causes generation of apoferritin in the cells of the reticuloendothelial system (Worwood, M., Iron in Biochemistry and Medicine, Volume II, Academic Press, 224-233 (1980) and Herbert V., J. Am. Diet Assoc., 92:1502-1509 (1992)). It is important that, although the literature almost exclusively uses the word "ferritin", what is generated within cells in response to an iron challenge is apoferritin, free of iron, which can then bind iron that would otherwise be cell-damaging. It binds and stores iron and prevents its uncontrolled release. The iron in it is in an iron core of ferric-oxide phosphate and, when the core is fully saturated, may consist of over 20% iron. About 2/3 of the body storage iron is in ferritin, with the remaining storage iron contained in hemosiderin, a denatured ferritin. One ferritin molecule is capable of binding up to a maximum of 4500 atoms of iron.
Ferritin which contains iron is called holoferritin. Holoferritin is not an acute phase reactant and, unlike apoferritin, the number of holoferritin molecules remains constant in the presence of inflammation or disease. What ferritin actually means, therefore, is the total of both apoferritin and holoferritin.
Circulating ferritin co-exists with serum transferrin (Tf) which is an iron delivery protein, for which there are receptors on every cell which needs iron. Transferrin absorbs iron from foodstuffs and delivers it to all cells which need iron as part of their normal metabolism. An example is red blood cells which need iron for the heme group of hemoglobin. Another example is muscle cells which use iron for the production of myoglobin. Tf delivers excess iron to storage cells which encapsulate the iron into holoferritin. Tf is a reverse acute phase reactant which means that in response to a disease state or inflammation the number of Tf molecules is reduced.
The holoferritin molecule can be analogized to a porous golf ball within which ferric iron is contained and is unable to escape. However, when ferric iron is reduced to ferrous iron by, for example, vitamin C or another reducing agent, ferrous iron is able to escape the ferritin molecule and is released into the circulation to generate harmful free radicals.
A diagnostic test for measuring total body tissue iron stores is important for diagnosing iron status, since either positive or negative iron balance promotes disease, susceptibility to disease, and aids a medical professional in diagnosing a patient and instructing the patient on the type of diet to maintain, in terms of iron content and iron availability for absorption. This is critical for those heterozygous for a gene promoting enhanced absorption, which can result in iron overload. Over 10% of Americans have an HLA-linked gene for enhanced iron absorption and about 30% of Africans have a non-HLA linked gene for enhanced iron absorption. Thus, in the United States, there is a large population at risk that is not identifiable by HLA typing, but is identifiable by the method of the present invention.
In people with genetically enhanced iron absorption, vitamin C supplements can only do harm. Vitamin C not only enhances iron absorption, but, worse, both releases catalytic iron from ferritin and drives the repetitive reduction of ferric to ferrous iron, with repetitive generation of more and more free radicals.
Current tests available for measuring total tissue iron stores are inaccurate because they only measure the number of molecules of ferritin protein, both apoferritin and holoferritin combined, and provide no information as to how much iron that ferritin protein carries. This is a crucial flaw since ferritin can carry no iron or up to 4500 atoms of iron. By convention, measurement of total ferritin protein is referred to as measurement of serum "ferritin".
In patient's with no illness or inflammation, "serum ferritin" generally provides an accurate reflection of body storage iron. A medical professional can measure the isolated total serum ferritin (.mu.g/L) in normal patients, multiply by ten, and determine total body tissue iron stores in grams. For example, the average ferritin (.mu.g/L) in serum in normal adult males is 100, 100.times.10=1000 mg, which is the average adult male total body tissue iron store. The average ferritin (.mu.g/L) in serum in adult females in the child-bearing years is 30, 30.times.10=300 mg, which is the average adult female (child-bearing years) body iron store. The female has 1/3 the body storage iron of the male because iron is lost monthly during menses.
Other standard serum iron measurements measure only the iron on Tf, which sticks out like a lollipop on the surface of the Tf molecule, plus low molecular weight iron (Borg, D. C., Handbook of Free Radicals and Antioxidants in Biomedicine, Volume I, CRC Press, Inc., 63-80 (1989)) such as might have been absorbed from iron succinate citrate (Aisen, P. et al., Int. Rev. Exp. Path., 31:1-46 (1990)). Standard serum iron measurements do not measure the iron on ferritin, since that iron is buried in the core of the ferritin molecule. In this test, therefore, what is being measured is the iron that is being delivered to cells which need iron, since Tf is an iron-delivery protein. This elucidates no information about the stores of iron in the body.
Another test used is the measurement of stainable iron in the bone marrow or quantitative measurement of liver iron, which is the "gold standard" for assessing body iron stores. This method is impractical for routine screening due to it being an invasive test. Further, serum iron and iron binding capacity (Tf) measurements together with the resultant percent saturation of the iron binding capacity, e.g. of Tf, have been used, but are unreliable because they are affected by diurnal variation, and by any inflammation since Tf is a reverse acute phase reactant, and by drug ingestion such as vitamin C or alcohol. Accordingly, the present tests used to measure total body tissue iron stores are prone to inaccurate readings.
An example which highlights the drawbacks of the current tests is a measurement of total ferritin isolated from a serum sample in patients with inflammation or disease. Using the current tests, when there is acute serum apoferritin elevation in response to inflammation or cancer, the serum ferritin level does not closely mirror body iron stores such that multiplying the serum ferritin in .mu.g/L by 10 gives a false reading with respect to the body iron stores in mg (Herbert V., et al., Stem Cells, 92:1502-1509 (1994)). Measurement of serum ferritin levels is generally accepted as the best non-invasive means of indirectly determining body iron stores only when the serum level of ferritin and the serum level of iron run in the same direction. This is because serum ferritin almost never accurately reflects body iron stores in any sick person, because serum ferritin=apoferritin+holoferritin, and, in any cell, reaction to illness or inflammation, for example, flu, pneumonia, rheumatoid arthritis, tonsillitis, heart disease, neoplastic diseases, renal failure or cancer, apoferritin rises, and iron quantity does not, and a reading of total ferritin provides inaccurate results.
Accordingly, measuring serum ferritin levels to determine total body tissue iron stores will lead to false results in a number of situations. A report that someone has a serum "ferritin" of 200 .mu.g/l, for example, is meaningless with respect to the iron content of that ferritin. Some of the "ferritin" may be pure apoferritin with no iron, some may be holoferritin saturated with 4,500 atoms of iron per molecule of ferritin, and most of the holoferritin is probably somewhere between no iron content and 4,500 atoms of iron per ferritin molecule,
In another example, serum "ferritin" levels in excess of 400 ng/ml are erroneously reported as defining iron overload by most clinical laboratories. Elevations in serum "ferritin" levels occur unrelated to iron stores in inflammatory conditions and in individuals with liver disease. In these situations serum "ferritin" is usually in excess of 400 ng/ml due to high apoferritin with no iron. Accordingly, the patient is diagnosed as having an iron overload when in reality there is only an apoferritin overload and iron levels may very well be normal. Further complicating the problem, many women ingest iron supplements, and this supplemental iron use has made iron/breast cancer risk studies especially difficult.
In a recent study (Adams, P. C., Amer. J. of Hemat., 45:146-149 (1994)) the usefulness of serum ferritin, Tf iron and Tf saturation were evaluated for the detection of patients heterozygous for hemochromatosis versus normals. In heterozygotes, hepatic iron is 3 to 4 fold increased over normals. The whole group of heterozygotes had a significantly increased mean serum ferritin and Tf saturation compared with the whole group of controls. However, only 11% of individual heterozygotes had elevated serum ferritin levels and only 8.6% had elevated Tf saturation. Thus, currently available tests miss 90% of those at risk. Heterozygotes are at increased risk of developing cancer and heart attacks, especially in the presence of substances which release iron from ferritin, such as alcohol and/or vitamin C supplements. Over 40% of Americans take vitamin C supplements.